PROJECT SUMMARY Interstrand DNA crosslinks are profoundly bioactive lesions because they block strand separation that is critical for most DNA transactions. ICLs are generated by a number of abundant endogenous metabolites, including abasic (AP) sites, environmental toxins, and anticancer drugs. Repair of ICLs is critical for genome stability and drug resistance. The mechanisms of ICL repair are not well understood, but the classical view of this process involves ICL ?unhooking? by nucleotide excision repair (NER) factors. Recently, however, a new base excision repair (BER) mediated pathway was discovered in two independent systems, whereby a DNA glycosylase unhooks the ICL through hydrolysis of the N-glycosidic bond, leaving the DNA backbone intact. The vertebrate specific DNA glycosylase NEIL3 unhooks AP- and psoralen-derived ICLs during DNA replication. In separate studies, a new DNA glycosylase (AlkZ) was identified in bacteria to unhook ICLs derived from the secondary metabolite azinomycin B, thus providing the producing organism with self-protection against this potent antimicrobial and antitumor agent. AlkZ-type proteins are abundant in bacterial pathogens known to produce toxins that cause cancer and other disorders. DNA glycosylases generally initiate base excision repair (BER) of small, modified nucleobases by ?flipping? the damaged base out of the double helix into the active site of the protein. Such a base-flipping mechanism is completely precluded for cross-linked bases and thus the structural mechanisms by which the recently discovered ICL-glycosylases operate are completely unknown and unprecedented. Given the importance of ICLs in human health, it is critical that we understand these unprecedented mechanisms of glycosylase-mediated ICL repair. The long term goals of this research are to elucidate the interplay between BER and NER pathways of ICL repair, and the role of DNA repair in pathogenicity of toxin producing bacteria in humans. The short term goals are to determine the molecular mechanisms of ICL unhooking by NEIL3 (Aim 1) and AlkZ (Aim 2). Our studies of two structurally unrelated proteins will reveal two specific unhooking mechanisms and, more importantly, will provide a broader general understanding of the fundamental chemical and structural strategies that have evolved for catalysis of ICL unhooking. This work will be carried out by an exceptional group of scientists focused on ICL repair using structural, chemical, and cellular approaches. The PIs and collaborators associated with this proposal are responsible for discovery and characterization of AP-derived ICLs and the NEIL3-dependent ICL repair pathway, production of stable ICLs suitable for structural and biochemical studies, and discovery of a non-canonical DNA glycosylase catalytic mechanism that enables repair of bulky DNA damage derived from complex secondary metabolites.